The present invention is a method for reproducing coniferous plants by somatic embryogenesis using the techniques of plant tissue culture. More specifically, it relates to the use of particular mixtures of growth hormones in the culture media used during the various stages of somatic embryo development. The invention is especially suited for producing large clones of superior selections useful for reforestation.
Loblolly pine (Pinus taeda L.), its closely related southern pines, and Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) are probably the most important commercial species of temperate North American timber trees. Similarly, Norway spruce (Picea abies (L.) Karst.) is probably the most important European softwood species. Since the early 1940s, when serious private reforestation efforts began, literally billions of one and two year old nursery-grown trees have been planted on cutover or burned forest lands. For many years these seedling trees were grown using naturally produced seed from cones collected as a part time effort of individuals seeking to supplement their incomes. As early as 1957 forest geneticists began to plant seed orchards using either seed or grafted scions obtained from superior trees discovered in the forests. These trees were selected for such inheritable characteristics as rapid growth, straightness of bole, wood density, etc. Now in both the southern pine and Douglas-fir regions the bulk of the seed is produced from selected trees grown in seed orchards, some of them now second and third generation orchards.
Despite the fact that the orchards were stocked with superior trees, pollination often cannot be carefully controlled and frequently the seed trees are fertilized by wild pollen of unknown characteristics. For this reason, the characteristics of the progeny produced by sexual reproduction have not been as predictable as hoped and genetic gain could not be attained as rapidly as desired.
Beginning about 1960, techniques were developed for reproducing some species of plants by tissue culture. These were predominately angiosperms and usually ornamental house plants. The method employed use of a suitable explant or donor tissue from a desirable plant. This was placed on a series of culture media in which nutrients and growth hormones were carefully controlled from step to step. The usual progression was growth from the explant to a callus. The callus was placed on a budding medium where adventitious buds formed. These, in turn, were separated, elongated, and rooted to ultimately form plantlets. A plantlet has the nature of a seedling but is genetically identical to the explant donor plant.
Gymnosperms in general, and most forest tree species in particular, proved to be much more difficult to reproduce by tissue culture. It was not until about 1975 that Douglas-fir was successfully reproduced by organogenesis. Loblolly pine was successfully reproduced about two years later.
A brief review of some of the most important work relating to the present invention will follow. This is intended to be representative only and is not fully inclusive of all the work in the field. Literature citations in the text are given in abbreviated form. Reference should be made to the bibliography at the end of the specification for full citations of the literature noted herein.
Culture by organogenesis is tedious and expensive due to the large amount of delicate manual handling necessary. It was soon recognized that embryogenesis was potentially a much more desirable method from the standpoints of quantity of plantlets produced, cost, potential genetic gain, and much lower probability of mutations. Work on embryogenesis of forest species began in the late 1970s. U.S. Pat. No. 4,217,730 to El-Nil describes one early attempt at somatic embryogenesis of Douglas-fir. This approach was later set aside because advanced stage embryos and plantlets could not be readily obtained. However, other workers entered the field in increasing numbers and progress has been rapid even if it has not until the present time reached the commercial stage.
Our earlier U.S. Pat. Nos. 4,957,866, 5,034,326, 5,036,007, herein incorporated by reference, describe improved methods of conifer embryogenesis. These also include extensive reviews of the most closely related literature. In the methods described in all of these patents, late stage proembryos, defined as totipotent embryonic structures having at least about 100 cells, are transferred and further cultured in a cotyledonary embryo development medium containing abscisic acid (ABA) as an essential growth hormone. It appears to be highly desirable during this stage to gradually reduce the level of exogenous ABA so that little or none is ultimately present. Other growth hormones; e.g. auxins, cytokinins, and gibberellins were not used at this time. The ultimate product of this culturing step is somatic embryos resembling natural mature zygotic embryos in morphology.
It is well accepted that plant tissue culture is a highly unpredictable science. Sondahl et al., in published European Patent Application 293,598, speak directly to this point.
"Since each plant species appears to possess a unique optimal set of media requirements, the successful preparation and regeneration of a new species cannot be necessarily inferred from the successful regimens applied to unrelated plant species." PA0 "Despite progress, our knowledge of embryogenesis is still fragmentary. At present we cannot yet define the conditions necessary for embryogenesis . . . "
This statement can be carried even farther. Rangaswamy (1986) notes that the potential for embryogenesis is even genotype specific within any given species.
Composition of the media used to initiate embryogenesis and induce embryo maturation are critical to success, regardless of the species being propagated. In particular, the type and level of the nitrogen source in the media and the presence or absence, composition, level, and timing of availability of growth hormones have been key to success. It is also these very factors, particularly the hormones, that have proved to be so unpredictable. As one example, Ammirato (1977), conducted a study examining the effects of zeatin (a cytokinin), ABA, and gibberellic acid (GA.sub.3) on the yield and morphology of caraway (Carum carvi) somatic embryos. These hormones were present singly and in all possible combinations in the media used for the later stages of embryo development. He concluded that a change in level or presence/absence of any one of the hormones caused a ripple effect felt throughout the system due to unpredictable interactions between the various hormones. The same problem is again discussed by Evans (1984) who notes that growth hormones which affect the same process can either act independently or may interact in some fashion.
The Ammirato (1977) paper is midrange in time between the first successful plant embryogenesis and the present. Much has been learned since then. However, this paper is useful in its clear and still valid presentation and characterization of the various growth hormones as promoters (or stimulators) and inhibitors. Evans (1984) once again expands Ammiratto's discussion. Auxins are seen by these investigators as promoting cell elongation, especially in shoot tissues, and in lower concentrations, in roots. Gibberellic acid (GA.sub.3) also promotes cell elongation in shoots but is either without effect or inhibitory in root tissues. ABA and ethylene are seen as inhibitors and tend to counteract the promotive effects of auxins and GA. Cytokinins appear to be more difficult to characterize. They generally tend to inhibit auxin induced cell elongation in stem and root tissues but act as promoters of leaf cell expansion.
In general, as far as conifer species are concerned, it appears that at least one exogenous auxin and usually a cytokinin are necessary hormones in a medium for the initiation of embryogenesis. Exogenous ABA is normally not used at this point nor is gibberellic acid (GA.sub.3) or its related gibberellins. The concentration of the growth hormones used in the initiation medium is typically then reduced or they are removed entirely as embryo development proceeds. However, auxins in particular may be beneficial at the stage of cotyledonary embryo development.
In most cases gibberellic acid appears to suppress embryogenesis; e.g., Kochba et al. (1978), Tisserat et al. (1977), Rajasekaran et al. (1987), Rangaswamy (1986). However, there are certainly exceptions. Chalupa (1990a, 1990b) refers to induction of embryogenesis in two Quercus species on a medium containing the cytokinin N.sup.6 -benzylaminopurine and gibberellic acid. Gmitter et al. (1990) appear to require gibberellic acid in their initiation medium for selected triploid hybrid citrus plants. Lakshmi Sita (1985) summarizes her earlier work and that of others in promoting embryogenesis of sandalwood (Santalum sp.). Gibberellic Acid was found to be useful in inducing embryogenesis using shoot explants in either solid or liquid suspension cultures. Despite her success, which included successful production of converted plants, she again points to the lack of predictability of embryogenesis.
Gibberellic acid has more frequently been used to promote late stage somatic embryo development and germination; e.g., Cruz et al. (1990), Eapen et al. (1990), Ghosh et al. (1991), Manrique et al. (1987), Trolinder et al. (1988). Nolan et al. (1988) and Garcia-Maya et al. (1990) note that gibberellins are involved in the synthesis of .alpha.-amylase. This enzyme is necessary for conversion of starches into sugars during germination.
Much less frequently various gibberellins have been used in combination with ABA at the late stage of embryo development and for stimulating germination; e.g., Eapen et al. (1989), Ferreira et al. (1990), Sondahl et al. (1988). Noriega et al. (1991) describe the first successful regeneration of hybrid tea rose by embryogenesis. Among the various media employed is an embryo maturation medium containing low concentrations of GA.sub.3 and ABA. Ammirato (1977), working with caraway (Carum carvi L.), reported that the beneficial effects of ABA on embryo maturation could be enhanced by gibberellic acid (GA.sub.3). However, in an extension of this work investigating the effect of mode of agitation, he noted that "the addition of GA.sub.3, alone or with ABA, had little effect on these cultures" (Ammirato (1983)). All of the reported work with this particular hormone combination has been exclusively with various angiosperm species.
The present inventors are aware of only one reported instance in which gibberellic acid has been examined in conifer somatic embryogenesis. Hakman and von Arnold (1985) tried adding ABA, IAA (indoleacetic acid), GA.sub.3, GA.sub.4/7, and activated charcoal singly to media used for further development of Picea abies (Norway spruce) proembryos. All of these were found to be ineffective and only a cytokinin-containing medium was useful under the conditions they employed.
Techniques to promote embryogenesis of numerous conifer genera are now well established. Research emphasis is now shifting to development of ways to scale up laboratory knowledge and techniques so that the process may become field operational on large scale. Yet many problems of a relatively fundamental nature still remain to be solved. One of these is improving somatic embryo quality and vigor. This is necessary so that germination to hardy plantlets and ultimate conversion to growing trees can be achieved at much higher percentages than has heretofore been possible. The present invention is directed to this end.